1、December 2010 Translation by DIN-Sprachendienst.English price group 10No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).I
2、CS 07.100.30; 67.120.10!$lW“1735952www.din.deDDIN EN ISO 13720Meat and meat products Enumeration of presumptive spp. (ISO 13720:2010)English translation of DIN EN ISO 13720:2010-12Fleisch und Fleischerzeugnisse Zhlung von prsumtiven spp. (ISO 13720:2010)Englische bersetzung von DIN EN ISO 13720:2010
3、-12Viande et produits base de viande Dnombrement des spp. prsomptifs (ISO 13720:2010)Traduction anglaise de DIN EN ISO 13720:2010-12SupersedesDIN ISO 13720:1998-05www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authoritative.1412.10 PseudomonasP
4、seudomonasPseudomonasDIN EN ISO 13720:2010-12 2 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food products”, Subcommittee SC 9 “Microbiology” (Secretariat: AFNOR, France) in collaboration with Technical Committee CEN/TC 27
5、5 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-01-06 AA Mikrobiologische Leb
6、ensmitteluntersuchung einschlielich Schnellverfahren. The DIN Standards corresponding to the International Standards preferred to in Clause 2 of the EN are as follows: ISO 6887-1 DIN EN ISO 6887-1 ISO 6887-2 DIN EN ISO 6887-2 ISO 7218 DIN EN ISO 7218 ISO/TS 11133-1 DIN ISO/TS 11133-1 ISO/TS 11133-2
7、DIN ISO/TS 11133-2 Amendments This standard differs from DIN ISO 13720:1998-05 as follows: a) only the oxidase test is now used for confirmation (inoculation into Kliglers agar has been deleted); b) the standard covers resumptive Pseudomonas spp.; c) the standard has been editorially revised. Previo
8、us editions DIN ISO 13720: 1998-05 pDIN EN ISO 13720:2010-12 3 National Annex NA (informative) Bibliography DIN EN ISO 6887-1, Microbiology of food and animal feeding stuffs Preparation of the test samples, of initial suspension and of decimal dilutions for microbiological examination Part 1: Genera
9、l rules for the preparation of the initial suspension and decimal dilutions DIN EN ISO 6887-2, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and decimal dilutions for microbiological examination Part 2: Specific rules for the preparation of meat and m
10、eat products DIN EN ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations DIN ISO/TS 11133-1, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 1: General guidelines on q
11、uality assurance for the preparation of culture media in the laboratory DIN ISO/TS 11133-2, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 2: Practical guidelines on performance testing of culture media DIN EN ISO 13720:2010-12 4 This pa
12、ge is intentionally blank EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 13720 September 2010 ICS 07.100.30; 67.120.10 English Version Meat and meat products - Enumeration of presumptive Pseudomonas spp. (ISO 13720:2010) Viande et produits base de viande - Dnombrement des Pseudomonas spp.
13、prsomptifs (ISO 13720:2010) Fleisch und Fleischerzeugnisse - Zhlung von prsumtiven Pseudomonas spp. (ISO 13720:2010) This European Standard was approved by CEN on 20 February 2010. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving thi
14、s European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN Management Centre or to any CEN member. This European Standard exists in three official versions
15、(English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bul
16、garia, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FO
17、R STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 13720:2010: EContents EN ISO 13720:2010 (
18、E) DIN EN ISO 13720:2010-12 2 Page Foreword3 1 Scope 4 2 Normative references 4 3 Terms and definitions .4 4 Principle4 5 Diluent, culture medium and reagent 5 6 Apparatus .7 7 Sampling.8 8 Preparation of test sample8 9 Procedure .8 9.1 Test portion, initial suspension and dilutions 8 9.2 Inoculatio
19、n and incubation .8 9.3 Counting and selection of colonies.9 9.4 Confirmation.9 9.4.1 Oxidase reaction9 9.4.2 Interpretation9 10 Expression of results 9 11 Test report 9 Bibliography 10 Foreword The text of ISO 13720:2010 has been prepared by Technical Committee ISO/TC 34 Food products” of the Inter
20、national Organization for Standardization (ISO) and has been taken over as EN ISO 13720:2010 by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” the secretariat of which is held This European Standard shall be given the status of a national standard, either by publication of an iden
21、tical text or by endorsement, at the latest by March 2011, and conflicting national standards shall be withdrawn at the latest by March 2011. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held res
22、ponsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Fin
23、land, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 13720:2010 has been approved by CEN as a EN
24、 ISO 13720:2010 without any modification. by DIN. “EN ISO 13720:2010 (E) DIN EN ISO 13720:2010-12 3 1 Scope This International Standard specifies a method for the enumeration of presumptive Pseudomonas spp. present in meat and meat products, including poultry. 2 Normative references The following re
25、ferenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 6887-1, Microbiology of food and animal feeding stuffs Prepar
26、ation of test samples, initial suspension and decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutions ISO 6887-2, Microbiology of food and animal feeding stuffs Preparation of test samples, initial suspension and dec
27、imal dilutions for microbiological examination Part 2: Specific rules for the preparation of meat and meat products ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations ISO/TS 11133-1, Microbiology of food and animal feeding stuf
28、fs Guidelines on preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory ISO/TS 11133-2, Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media Part 2: Practic
29、al guidelines on performance testing of culture media 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 presumptive Pseudomonas spp. bacteria which at 25 C form colonies in cephalothin-sodium fusidate-cetrimide (CFC) agar and which show a posit
30、ive oxidase reaction when tested according to the method described in this International Standard 4 Principle An initial suspension and decimal dilutions are prepared from the test sample. The solid selective medium, CFC agar, is inoculated with a specified quantity of the initial suspension of the
31、product. EN ISO 13720:2010 (E) DIN EN ISO 13720:2010-12 4 Other plates are prepared under the same conditions, using decimal dilutions of the initial suspension. The plates are incubated at 25 C for 44 h 4 h. Colonies of presumptive Pseudomonas spp. are confirmed by the oxidase test (positive). The
32、number of presumptive Pseudomonas spp. per millilitre, or per gram, of the test sample is calculated from the number of confirmed colonies per plate. 5 Diluent, culture medium and reagent 5.1 General For current laboratory practice, see ISO 7218; for preparation and testing of media, see ISO/TS 1113
33、3-1 and ISO/TS 11133-2. 5.2 Diluent See ISO 6887-1 and ISO 6887-2. 5.3 Cephalothin-sodium fusidate-cetrimide agar (see Reference 3) 5.3.1 Basic medium 5.3.1.1 Composition Enzymatic digest of gelatin 16,0 g Enzymatic digest of casein 10,0 g Potassium sulfate (K2SO4) 10,0 Magnesium chloride (MgCl2) 1,
34、4 g Agara12,0 g to 18,0 gWater 1 000 ml aThe mass used depends on the gel strength of the agar. 5.3.1.2 Preparation Dissolve the basic components or the dehydrated basic medium in the water, by bringing to the boil. Adjust the pH (6.4), if necessary, so that after sterilization it is 7,2 0,2 at 25 C
35、. Dispense the basic medium into flasks or bottles of suitable capacity (6.6). Sterilize for 15 min in the autoclave (6.1) at 121 C. 5.3.2 Inhibitor solutions Do not keep solutions for more than 7 days at 5 C 3 C. 5.3.2.1 Cephalothin solution 5.3.2.1.1 Composition Cephalothin sodium salt 0,1 g Water
36、 100 ml EN ISO 13720:2010 (E) DIN EN ISO 13720:2010-12 5 5.3.2.1.2 Preparation Dissolve the cephalothin in the water and sterilize the solution by filtration. 5.3.2.2 Sodium fusidate solution 5.3.2.2.1 Composition Sodium fusidate 0,1 g Water 100 ml 5.3.2.2.2 Preparation Dissolve the sodium fusidate
37、in the water and sterilize the solution by filtration. 5.3.2.3 Cetrimide solution 5.3.2.3.1 Composition Cetrimidea0,1 g Water 100 ml aMixture consisting chiefly of tetradecyltrimethylammonium bromide together with smaller amounts of dodecyltrimethylammonium bromide and cetrimonium (hexadecyltrimethy
38、lammonium) bromide. 5.3.2.3.2 Preparation Dissolve the cetrimide in the water and sterilize the solution by filtration. 5.3.3 Complete medium 5.3.3.1 Composition Volume ml Final concentration g/ml Basic medium (5.3.1) 100 Cephalothin solution (5.3.2.1) 5 50 Sodium fusidate solution (5.3.2.2) 1 10 Ce
39、trimide solution (5.3.2.3) 1 10 5.3.3.2 Preparation Add the inhibitor solutions to the basic medium, cooled in a water bath (6.3) to 47 C 2 C, then mix carefully. 5.3.4 Preparation of CFC agar plates Pour amounts of approximately 15 ml of the complete medium (5.3.3) into sterile Petri dishes (6.8) a
40、nd allow to solidify. Immediately before use, the agar plates should be dried in accordance with ISO/TS 11133-1. If prepared in advance, the undried agar plates shall be kept for not more than 4 weeks at 5 C 3 C. EN ISO 13720:2010 (E) DIN EN ISO 13720:2010-12 6 5.3.5 Performance testing For the defi
41、nition of selectivity and productivity refer to ISO/TS 11133-1. The performance of CFC agar shall be tested according to the methods and criteria described in ISO/TS 11133-2. 5.3.5.1 Productivity Incubation: At 25 C 1 C for 44 h 4 h Strain: Pseudomonas fluorescens WDCM 001151)or Pseudomonas fragi WD
42、CM 001161)Reference medium: Soybean-casein digest agar medium (TSA) Method of control: Quantitative Criteria: Productivity ratio PRW 0,5 5.3.5.2 Selectivity Incubation: At 25 C 1 C for 44 h 4 h Strain: Escherichia coli WDCM 000131)Method of control: Quantitative Criteria: Total inhibition 5.4 Reagen
43、t for the detection of oxidase 5.4.1 Composition N,N,N,N-Tetramethyl-p-phenylenediamine dihydrochloride 1,0 g Water 100 ml 5.4.2 Preparation Dissolve the reagent in the water immediately before use. Commercially available discs or sticks may be used. In this case, follow the manufacturers recommenda
44、tions. 6 Apparatus Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following. 6.1 Oven for dry sterilization or autoclave for wet sterilization. 6.2 Incubator, capable of operating at 25 C 1 C. 1) Refer to the reference strain catalogue available (viewed 2010-07-19)
45、 on http:/www.wfcc.nig.ac.jp/WDCM_Reference_Strain_Catalogue for information on culture collection strain numbers and contact details EN ISO 13720:2010 (E) DIN EN ISO 13720:2010-12 7 6.3 Water bath, capable of operating at 47 C 2 C. 6.4 pH-meter, capable of measurements to an accuracy of 0,05 pH uni
46、ts. 6.5 Loops made of platinum-iridium alloy or equivalent sterile disposable loops. 6.6 Test tubes, bottles or flasks, of appropriate capacity. 6.7 Total-delivery pipettes, sterile, of nominal capacity 1 ml and graduated in 0,1 ml divisions, ISO 8351class A, or automatic pipettes, ISO 8655-22, with
47、 use of sterile tips. 6.8 Petri dishes, made of glass or plastic, of diameter 90 mm to 100 mm. 6.9 Spreaders, made of glass or plastic, e.g. hockey sticks made from a glass rod of approximately 3,5 mm diameter and 200 mm length, bent at right angles about 30 mm from one end and with the cut ends ann
48、ealed. 7 Sampling Sampling is not part of the method specified in this International Standard. If there is no specific International Standard dealing with sampling of the product concerned, it is recommended that the interested parties come to an agreement on this subject. It is important that the l
49、aboratory receive a sample which is truly representative and which has not been damaged or changed during transport or storage (see ISO 7218). 8 Preparation of test sample Prepare the test sample in accordance with ISO 6887-1 and ISO 6887-2 and/or the specific International Standard appropriate to the product concerned. If there is no specific International Standard, it is recommended that the parties concerned come to an agreement on this subject. 9 Procedure 9.1
